After-cooking darkening (ACD) is a non-enzymatic gray-black discoloration of potato tuber flesh occurring after cooking. The discoloration is due to the formation of a colorless iron-chlorogenic acid complex during the cooking process, which upon exposure to air, oxidizes to form the dark ferri-dichlorogenic acid (Dale and Mackay, 1994). To prevent the discoloration caused by ACD, processors in the French fry industry treat the French fried potato strips with sodium acid pyrophosphate (SAPP, Na2H2P7O7). Sodium acid pyrophosphate reduces darkening by complexing with the iron in the tuber. In this capacity the iron is held in a nonionizable form and cannot take part in the reaction with chlorogenic acid (Smith, 1987). A rise in the number of French fry processing industries has led to an increase in SAPP usage. The phosphorus residue released from SAPP during processing, has made it mandatory to eliminate SAPP from industrial wastewater. This currently involves the removal of phosphorous from wastewater through chemical precipitation, adding further to processing costs for the French fry industry (Wang-Pruski and Nowak, 2004). Considering the millions of dollars per year that SAPP costs the industry, it would be beneficial both from economical and environmental standpoints to reduce or eliminate the use of SAPP in the processing industry.
Traditional breeding has led to the development of many low-ACD cultivars, including the cultivars Red Pontiac and Yukon Gold. However, cultivars for French fry production must also possess traits essential for processing, specifically oblong tuber shape, shallow eyes, high specific gravity, low reducing sugars, high yield, and resistance to diseases (Bradshaw et al., 1998). Currently, Russet Burbank and Shepody are the primary cultivars used in the French fry processing industry in Canada. Both cultivars require the use of SAPP to prevent the darkening. To date no cultivars are available that possess all the traits essential for French fry processing, as well as complete resistance to ACD (Wang-Pruski, personal communication).
Chlorogenic acid (CgA) is not only involved in ACD but it also has various biological roles, including the involvement in defense mechanisms against insects or phytopathogens, disease and fungal resistance, growth regulation, and wound response (Kühnl et al., 1987; Yao et al., 1995; Friedman, 1997; Griffiths and Bain, 1997). In potatoes specifically, CgA is able to provide covalent cross-links between polysaccharides and cell well proteins; making the cell wall stronger and more resistant to invading pathogens (Yao et al., 1995). Once the threat (pathogen or disease) subsides, normal oxidative processes lower the accumulated CgA in suberized tissues (Friedman, 1997). Chlorogenic acid accounts for up to 90% of the total phenolic compounds present in the potato tuber (Griffiths and Bain, 1997; Lewis et al., 1998; Lugasi et al., 1999; Percival and Baird, 2000). Approximately 50% of the CgA is located in the potato peel and adjoining tissues. Chlorogenic acid is synthesized via the phenylpropanoid pathway, which has not been explored in great detail, especially in species from the Solanaceae family.
Cinnamic acid 4-hydroxylase (C4H, EC 1.14.13.11) is an essential enzyme for the biosynthesis of CgA and therefore is thought to play a key role in the ACD mechanism. Cinnamic acid 4-hydroxylase catalyzes the hydroxylation of t-cinnamic acid to form p-coumaric acid, during the synthesis of CA. The C4H enzyme belongs to the CYP73 family of plant cytochrome P450 proteins. C4H enzymatic activity is induced by wounding, light, and pathogen infection in various plant species (Tanaka et al., 1974; Fahrendorf and Dixon, 1993; Bell-Lelong et al., 1997; Petersen, 2003). Class I and II forms of the gene encoding C4H have been sequenced in many plant species, including Arabidopsis, Jerusalem artichoke, red pepper, pea, alfalfa, and species of Populus and Citrus. Class I c4h is the predominate form found in almost all plant species, whereas the divergent class II form has only been isolated from orange and French bean. The divergent class II c4h has approximately 60% sequence similarity to the class I form and differs in the N-terminus and three internal domains (Betz et al., 2001; Blee et al., 2001).
The gene expression level of c4h depends on the specific plant species, tissue type, as well as stress and environmental factors (Whitbred and Schuler, 2000). Bell-Lelong et al. (1997) and Mizutani et al. (1997) found that in Arabidopsis, c4h was expressed in all tissues analyzed including leaves, seedlings, stems, flowers, and roots. The higher levels were found in the stems and roots, possibly because of C4H's role in the production of the monolignols required for lignification. The c4h gene has not been sequenced nor has its expression profile been identified in potato. To date, no genes in potato have been identified that relate to the control of ACD.